Rapid quantitative measurement of soluble fibrin in opaque body fluids

ABSTRACT

A method for determining the existence and the amount of soluble fibrin contained in a specimen fluid is provided. The method includes the steps of precipitating soluble fibrin out of the opaque specimen fluid, aggregating the soluble fibrin precipitates in a limited region of a transparent container so as to render the precipitates optically detectable in the opaque specimen fluid, and optically detecting the precipitates. The amount of soluble fibrin may be determined by measuring the time from the addition of the precipitating regent to the detection of the soluble fibrin precipitates. Methods of the present invention allow one to measure soluble fibrin in whole blood, and therefore render the test useful in the operating room under conditions of major surgery and in the presence of severe trauma wherein DIC is likely to supervene.

CROSS REFERENCE TO RELATED ART

[0001] A commonly owned and issued patent number 5,716,796, entitled“Optical Blood Hemostatic Analysis Apparatus and Method” by Brian S.Bull and Ralph A. Korpman, filed on Jan. 29, 1996, is herebyincorporated in its entirety by reference.

[0002] This application is a continuation-in-part of the pendingapplication serial number 09/021,062, entitled “A Rapid QuantitativeMeasurement of Soluble Fibrin In Whole Blood,” filed on Feb. 9, 1998,the content of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] Area of the Art

[0004] The invention relates generally to optical blood hemostaticanalysis methods and specifically to methods for quantitativelymeasuring the concentration of soluble fibrin molecules in opaque bodyfluids such as whole blood.

DESCRIPTION OF THE PRIOR ART

[0005] Soluble fibrin (SF) molecules are generated from fibrinogenmolecules when thrombin cleaves the alpha and beta polypeptide chains ofthe fibrinogen molecules. The presence of SF molecules in the bloodstream is one of the most sensitive indicators of early disseminatedintravascular clotting (DIC). DIC poses a great threat to patientsduring major surgery and after severe trauma. Therefore, a technique forrapidly and quantitatively measuring levels of soluble fibrin in wholeblood would be very useful during major surgery and after severe trauma.

[0006] Although the measurement of SF has been carried out for manyyears, the measurement of soluble fibrin under the circumstances ofmajor surgery or severe trauma has always been problematic and thetechnique is seldomly used. All available techniques for SF measurementrequire that the procedure be run on plasma rather than on whole blood;such a procedure takes additional time. In addition, most availabletechniques are only semiquantitative (the results are in the form of(+—++++)). These problems have prevented the test from being widelyused. Available techniques must utilize plasma because the detection ofSF precipitates is done optically and a clear fluid medium is thus anecessity. In addition, in those techniques, optical endpoints aredesirable for tests of the coagulation process as they typically permitthe entire reaction chamber to be disposable. Optical endpoints for SFin whole blood are typically useless, however, owing to the opacity ofthe whole blood and the widely distributed nature of the SFprecipitates. Therefore, the previous techniques that utilize opticalendpoints require the use of plasma.

[0007] Plasma is prepared from whole blood by centrifugation. Thecentrifugation step typically requires the sample to be transported outof the operating room to an analytical laboratory. The minimum time fora test result thus becomes 30-45 minutes. Once the transport time hasbeen added to the time required for centrifugation and the time foranalysis, the test result is useless in the context of the operatingroom as the patient's condition would have likely changed substantiallyin the meantime. For studying the kinetics of SF conducting a test thatrequires preparation of plasma is likewise problematic as the reactionof thrombin on fibrinogen takes place on the order of seconds and cannotbe stopped without using anti-polymerizing agents that interfere withsubsequent detection of SF.

[0008] Most available methods for detection of SF quantify the amount ofSF present only in a very approximate manner as in +—++++. Those that doa more rigorous quantification require additional steps such as:

[0009] 1. removing the SF precipitates, drying and weighing theprecipitates;

[0010] 2. removing the SF precipitates, analyzing them for proteincontent;

[0011] 3. Immunologically precipitating the SF precipitates, using asecond antibody to quantify the amount.

[0012] All of these techniques add even more time to the SF analysis.They render the test less useful as a result.

[0013] Therefore, a need exists to develop a method that performs a SFtest in an opaque suspension such as whole blood. Such a method wouldhave far greater utility than presently available tests which, due totheir optical endpoints, must be performed in plasma. Indeed, it is onlywhole blood tests that can be utilized in an operating room where therapid measurement of SF will permit the surgeon or the anesthesiologistto take immediate steps to correct the conditions that are leadingtowards DIC.

SUMMARY OF THE INVENTION

[0014] It is an object of the present invention to provide a method forrapidly quantifying soluble fibrin optically in opaque suspensions ofwhole blood so as to render the test useful in the operating room underconditions of major surgery and in the presence of severe trauma whereDIC is likely to supervene.

[0015] These objectives and advantages are achieved by a method of thepresent invention. In accordance with the embodiments of the presentinvention, the method includes the steps of:

[0016] (a) mixing a portion of the opaque specimen fluid in atransparent container with a sufficient amount of precipitating reagentunder a condition that causes the soluble fibrin to precipitate;

[0017] (b) aggregating and concentrating the soluble fibrin precipitatesin a region of the container for rendering the precipitates opticallydetectable in the opaque specimen fluid;

[0018] (c) optically detecting the precipitates;

[0019] (d) recording the time when the precipitates are first becomeoptically detectable in the opaque specimen fluid, wherein the timeelapsed from the addition of the precipitating reagent to the detectionof the aggregated precipitates is an inverse measure of the quantity ofsoluble fibrin present in the opaque specimen fluid.

[0020] A method in accordance with the present invention provides anumber of advantages. As explained in greater detail below, the methodsof the present invention provide a rapid and quantitative measurement ofsoluble fibrin in opaque body fluids such as, but not limited to, wholeblood. Particularly, methods of the present invention make it possibleto use optical endpoints to measure SF in whole blood. It is known inthe art that optical endpoints are desirable for tests of thecoagulation process as they typically permit the entire reaction chamberto be disposable. However, prior to the present invention, opticalendpoints for SF in whole blood are typically useless owing to theopacity of the whole blood and the widely distributed nature of the SFprecipitates. The present invention allows SF to form precipitates andto be collected in a small, predictable portion of the reaction mixtureso that they can be readily detected optically. When collected in highconcentration, SF precipitates exclude the opaque whole blood medium andbecome optically detectable from outside the reaction chamber. Methodsof the present invention eliminate the plasma preparation steps, andprovide rapid and effective SF measurements that are readily usable inthe operating room under conditions of major surgery and in the presenceof severe trauma where DIC is likely to supervene. Since the presentinvention can accurately detect and measure the amount of soluble fibrinin an opaque specimen fluid, the present invention provides an effectivemeans to detect early disseminated intravascular clotting.

[0021] The methods of the present invention are well suited for useduring major surgery, after severe trauma, and similar circumstanceswhere disseminated intravascular clotting poses its greatest threat. Amethod in accordance with the present invention can effectively detectDIC by rapidly and quantitatively measuring the soluble fibrin.

[0022] The invention is defined in its fullest scope in the appendedclaims and is described below in its preferred embodiments.

DESCRIPTION OF THE FIGURES

[0023] The above-mentioned and other features of this invention and themanner of obtaining them will become more apparent, and will be bestunderstood by reference to the following description, taken inconjunction with the accompanying drawings. These drawings depict only atypical embodiment of the invention and do not therefore limit itsscope. They serve to add specificity and detail, in which:

[0024]FIGS. 1a and 1 b are diagrammatic side views of one embodiment ofthe invention, showing the two positions in the rocking motion of theapparatus. FIG. 1c is a diagrammatic view of the apparatus shown in FIG.1a.

[0025]FIG. 2 is a plot of the kinetics of the production of SF in wholeblood.

[0026]FIG. 3a shows a series of fibrinogen measurements performed onsamples from a patient undergoing liver transplantation.

[0027]FIG. 3b shows three other assays performed on the same patient:measurements of D-dimer, fibrin degradation products (FDP) and solublefibrin (SF) in relation to the time of liver replacement.

DETAILED DESCRIPTION OF THE INVENTION

[0028] One aspect of the present invention provides a method fordetermining the existence and the amount of soluble fibrin contained inan opaque specimen fluid. In accordance with embodiments of the presentinvention, the method comprises the steps of:

[0029] (a) mixing a portion of the opaque specimen fluid in atransparent container with a sufficient amount of precipitating reagentunder a condition that causes the soluble fibrin to precipitate;

[0030] (b) aggregating and concentrating the soluble fibrin precipitatesin a region of the container for rendering the precipitates opticallydetectable in the opaque specimen fluid;

[0031] (c) optically detecting the precipitates;

[0032] (d) recording the time when the precipitates are first becomeoptically detectable in the opaque specimen fluid, wherein the timeelapsed from the addition of the precipitating reagent to the detectionof the aggregated precipitates is an inverse measure of the quantity ofsoluble fibrin present in the opaque specimen fluid.

[0033] For purposes of the present invention, the opaque specimen fluidcan be any body fluids that contain soluble fibrin. The inventions are,however, particularly applicable to opaque body fluids such as wholeblood, bloody effusions, bloody cerebrospinal fluid and the like.Preferably the specimen fluid is whole blood. In one embodiment of thepresent invention, whole blood is diluted. The whole blood can bediluted by a diluent such as saline solution.

[0034] A precipitating reagent is any reagent which can cause solublefibrin to precipitate out of the soluble fibrin-containing specimenfluid. Examples of a precipitating reagent include, but are not limitedto, protamine sulfate, polybrene, and the like. Preferably, theprecipitate reagent is protamine sulfate.

[0035] The amount of a precipitating reagent is sufficient if it cancause substantially all of the soluble fibrin contained in a specimenfluid to precipitate out of the fluid. One skilled in the art canreadily determine the amount of the precipitating reagent that should beused without undue experimentation in view of the instant disclosure.

[0036] A portion of the opaque specimen fluid is mixed with aprecipitating reagent under a condition that causes the soluble fibrinto precipitate. In one embodiment of the present invention, the mixingmay take place at a pH of about 5.9 or below, and at a temperature ofabout 37° C.

[0037] According to embodiments of the present invention, the solublefibrin precipitates contained in a transparent container may beaggregated and concentrated to a limited region of the container byplacing the transparent container to an apparatus of the presentinvention. The apparatus used in the present invention must have thecharacteristics of producing in the reaction mixture hydraulic flowpatterns so that the apparatus will aggregate and concentrate the SFprecipitates in a highly localized portion of the reaction mixture so asto render them visible in an opaque medium such as diluted whole blood.In one embodiment of the present invention, an optical blood homeostaticanalysis apparatus as described in U.S. Pat. No. 5,184,188, the relevantcontent of which is incorporated herein in its entirety by reference.

[0038] In general, an optical blood hemostatic analysis apparatus iscapable of both rotating and rocking a transparent, approximatelycylindrical specimen container (for example, a 12×75 mm glass test tube)while maintaining the container in a nearly horizontal position and at atemperature of approximately 37° C. Specimen fluid and a precipitatereagent are introduced into the open end of the container, and are thenmixed and incubated by the apparatus.

[0039] As described in the '188 patent, one embodiment of the mechanicalapparatus of the invention is illustrated in FIGS. 1a and 1 b, whichshow a specimen container 1 containing specimen fluid 2, which issupported in a nearly horizontal position on a pair of rotating rollers3. FIG. 1c is an end-view of the apparatus shown in FIGS. 1a and 1 b.The rollers 3 may be rotated by any convenient means, such as anelectrical motor coupled to the rollers either directly or through adrive train (e.g., a belt, gears, or a friction drive). In the preferredembodiment, the specimen container 1 is rocked longitudinally around anapproximate mid-point pivot P through an angle of preferably about ±3°,as shown by comparing FIG. 1a with FIG. 1b. The rotational rate of thespecimen container 1 is typically about 12 rpm, and the rocking rate istypically about 15 cycles per minute. The rocking motion may be impartedby a cam or any other convenient means. The specific rocking androtation rates are not necessarily critical, but the particular valuesdescribed above have been found to be effective.

[0040] The specimen fluid 2 does not run out of the open end of asmall-diameter specimen container 1 when tilted forward because of thestrong surface tension of the contained fluid, aided by the small radiusof curvature of the rim of the container 1. During the rotating androcking, the precipitating reagent causes the soluble fibrin containedin the diluted whole blood to precipitate out of solution. As theprecipitation reaction is occurring, the rocking and rotating motionmoves the not-yet-visible fibrin monomer (SF) clumps to a region whereall of the SF in the entire sample will accumulate. As they reach thispreferred region the clumps interact with SF material already theremaking the location even more attractive for SF accumulations that arestill too small to be detected optically. If the amount of SF issufficiently large, the SF precipitates will adhere to the inner tubesurface and be lifted clear of the reaction mixture.

[0041] The time the precipitates first become optically detectable isdefined as the first end-point. The time the precipitates stick to thetube and rotate with the tube is defined as the second end-point. Thetime that it takes from the start of the process to the first end-point,and to the second-end point will be measured. The time measurements canbe correlated to the quantity of SF by a standard curve respectively.The SF standard curve is produced by adding known quantities of SF tonative whole blood and analyzing the resulting mixtures.

[0042] For the purpose of determining the quantity of SF in a specimenfluid, one may use either of the time measurements or both to determinethe quantity of SF with a standard curve. The time that it takes fromthe start of the process to the second-end point is easier to measure.However, when the level of SF is low, the first end-point may be theonly one available since the precipitates may not be concentrated enoughto reach the second end-point. In this case, it is preferred to use thetime measurement of the first end-point to determine the quantity of SF.Any comparable means of accomplishing this goal is within the scope ofthis invention, including simple rotation or agitation of a sealedcontainer, or use of a container having a liquid retention rim at itsopening.

[0043] Since the process works best at 37° C., it is desirable tomaintain the specimen at that temperature. The temperature of thespecimen fluid can be maintained, for example, by a tungsten filamentlight bulb 4 situated near the specimen container 1 and controlled by athermostatic circuit 5 having a temperature sensor 6 adjacent to or incontact with the specimen container 1. Alternatively, the temperaturescan be maintained by placing the specimen container 1 in an incubatedchamber having the desired temperature.

[0044] The time that it takes from the start of the process to theend-points can be measured by a variety of means that detect theend-points. A number of different types of end-point detectors can beused in the invention. Examples of types of end-point detectors include,but are not limited to, direct visual observation; a flying spotscanner; a charge couple device (CCD) camera, a detector consisting of anarrow beam of light (e.g., from a small, solid state laser) that iscaused to sweep (e.g., by means of an oscillating mirror) the inner wallof the transparent specimen container from a position just outside theopen end of the container; and a detector that uses a light beam and atime-delay discriminator circuit which distinguishes the presence ofsoluble fibrin precipitates from the rocking surface of the blooditself. The above detectors are fully described in the '188 patent, thecontent of which is incorporated herein by reference.

[0045] It is to be understood that the optical blood hemostatic analysisapparatus described above has been chosen only for the purpose ofdescribing a particular embodiment and function of the invention. Othertypes of optical apparatus may also be used as long as the apparatus canperform the same function as the one described above. Suitable apparatuswould be evident to those of ordinary skill in the art in view of thisdisclosure.

[0046] In one embodiment of the present invention, the measurement stepsare repeated with a second precipitating reagent. In accordance withthis embodiment, the measurement method further includes the steps ofmixing another portion of the opaque specimen fluid with a secondprecipitating reagent and repeating steps (a) to (d) described above toobtain a second timing measurement for elapsed time. Then the firsttiming measurement and the second timing measurement are related torespective standard curves prepared with respective first and secondprecipitating reagents. The quantity of soluble fibrin present in theopaque specimen sample is determined by an average of the twomeasurements. For the purpose of the present invention, the first andthe second precipitating reagents may be the same reagents at differentconcentrations or may be two different reagents.

[0047] It should be understood that portions of an opaque specimen fluidmay be mixed respectively with two or more different concentrations of aprecipitating reagent to obtain two or more measurements, and thequantity of SF contained in the specimen fluid may be determined byaveraging the measurements. Alternatively, portions of an opaquespecimen fluid may be mixed with two or more different types ofprecipitating reagents to achieve the same results.

EXAMPLE I The Kinetics of the Production of SF in Whole Blood

[0048] A method of determining soluble fibrin with the present inventionis used to measure the amount of SF in a whole blood sample treated withthrombin to determine the kinetics of the reaction. 2 ml of whole bloodwas mixed with 0.01 NIH units of thrombin at time zero and 150 μlaliquots were removed at the times noted. Each aliquot was suspended inabout 450 μl of saline solution at pH 5.0 and was placed in a specimencontainer together with about 20 μl of the precipitating reagent,protamine sulfate. The time of the appearance of precipitates wasmeasured and recorded in SF units. The results is summarized in Table I.TABLE I Time after Thrombin SF Time Soluble Visible Addition (min) (sec)Fibrin Units Fibrin 0 250 2.8 No 1 12.5 56.0 No 2 10.2 68.6 No 3 9.871.4 No 4 9.9 70.7 No 5 11.2 62.5 No 8 13.5 51.9 Yes 10 14.4 48.6 Yes 1914.7 47.6 Yes 31 17.0 41.2 Yes 70 19.4 36.1 Yes 112 20.2 35.0 Yes 18921.2 33.0 Yes

[0049]FIG. 2 is a plot of the kinetics of the production of SF in wholeblood. At the point marked by the arrow thrombin is added to thereaction mixture. The present invention is utilized to perform analysisat intervals of 1-30 minutes. Over an initial period of 3-4 minutesafter the addition of a small quantity of thrombin, the SF concentrationtiter rises. The early SF molecules produced can be carried by thefibrinogen and kept from polymerizing as a visible fibrin clot.Following a peak concentration reached at approximately three minutes,the SF present in excess of the carrying capacity of the fibrinogenprecipitates out of the mixture in the form of a visible fibrin clot andover the next 30 minutes the level of SF decreases by about 50%. Overthe ensuing hours, the SF level declines slightly but is essentiallystable for periods in excess of 2 hours.

[0050] As noted in FIG. 2, the kinetics of the reaction require a testthat can be completed within minutes, hence require that the test beperformed in whole blood.

EXAMPLE II SF Measurement in Whole Blood

[0051] In a second test of the system, a series of 150 μl whole bloodsamples were, at thirty minute intervals, removed from a patientundergoing liver transplantation. These samples were suspended in about450 μl saline, and were placed in a specimen container together withabout 20 μl of the precipitating reagent, protamine sulfate. The wholeblood sample was taken from a patient at the start of the operation,then every 30 minutes with samples spaced more closely during thecritical reperfusion phase of the transplantation process. The mixtureof protamine sulfate and whole blood was placed in a hemostatic analysisapparatus as described in the '188 patent at a temperature of about 37°C. to allow the mixture to react until precipitates formed and adheredto the inner tube surface. The time that it took from the start of theprocess to the first-end point, and the second end-point were thenmeasured, and the measurements were correlated to the quantity of SF bya standard curve.

[0052] Other portions of the same samples were centrifuged to recoverplasma and refrigerated until the next day so that additional assays forfibrin degradation products (FDPs), D-dimer levels and fibrinogen levelscould be performed. These four assays are plotted in FIGS. 3a and 3 b toshow the congruence of the results.

[0053]FIG. 3a is a series of fibrinogen measurements performed onsamples from a patient undergoing liver transplantation. FIG. 3b showsthree other assays performed on the same patient: measurements ofD-dimer, fibrin degradation products (FDPs) and soluble fibrin (SF) inrelation to the time of liver replacement. Only the SF tests wereperformed during the actual surgery. The remaining tests were performedon the following day since all of the remaining tests required thepreparation of plasma from the whole blood samples and the patient'scondition was changing so rapidly that the values were not useful to theoperating surgeon. The close correlation of the various measures on allfour test methods indicates that the information provide by the SF assaywithin minutes of the time the sample was drawn is useful for managing avery unstable patient.

[0054] Correlation coefficients are summarized in Table II. Table IIconfirms the visual impression that all four assays are highlycorrelated—that as fibrinogen disappears from the patient's circulationSF and the various measures of fibrinogen/fibrin breakdown (FDP,D-dimer) begin to appear. TABLE II Correlation coefficients (r) 0.9026SFU vs FDP dilution 0.8558 SFU vs D-Dimer dilution −0.8572 SFU vsFibrinogen

[0055] The foregoing is meant to illustrate, but not to limit, the scopeof the invention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

[0056] The present invention may be embodied in other specific formswithout departing from its essential characteristics. The describedembodiment is to be considered in all respects only as illustrative andnot as restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of the equivalence ofthe claims are to be embraced within their scope.

What is claimed is:
 1. A method for determining the existence and theamount of soluble fibrin contained in an opaque specimen fluid, themethod comprising the steps of: (a) mixing a portion of the opaquespecimen fluid in a transparent container with a sufficient amount ofprecipitating reagent under a condition to cause the soluble fibrin toprecipitate; (b) aggregating and concentrating the soluble fibrinprecipitates in a region of the container for rendering the precipitatesoptically detectable in the opaque specimen fluid, (c) opticallydetecting the precipitates, (d) recording the time when the precipitatesare first become optically detectable in the opaque specimen fluid,wherein the time elapsed from the addition of the precipitating reagentto the detection of the aggregated precipitates is an inverse measure ofthe quantity of soluble fibrin present in the opaque specimen fluid. 2.The method of claim 1 further comprising a step of recording the timewhen the precipitates stick to the container and rotate with thecontainer, and wherein the time elapsed from the addition of theprecipitating reagent to the detection of the sticking and rotatingaggregated precipitates is an inverse measure of the quantity of solublefibrin present in the opaque specimen fluid.
 3. The method of claim 1,wherein the step (b) further comprises the steps of (a) placing thetransparent container containing the mixture within an apparatus that iscapable of subjecting the container to both rocking and rotating motion;and (b) subjecting the mixture contained in the container placed withinthe apparatus to rocking and rotating motion to aggregate andconcentrate the soluble fibrin precipitates in a limited region of thecontainer.
 4. The method of claim 1 further comprising a step ofrelating the recorded time to a standard reference curve measured onsamples with a known soluble fibrin content to determine theconcentration of soluble fibrin contained in the opaque specimen fluid.5. The method of claim 1, wherein the precipitating reagent is a firstprecipitation reagent, and the time recorded in step (d) is a first timemeasurement, and the method of claim 1 further comprising the steps ofmixing another portion of the opaque specimen fluid with a secondprecipitating reagent and repeating steps (a) to (d) of claim 1 toobtain a second time measurement for elapsed time.
 6. The method ofclaim 5, wherein the first time measurement and the second timemeasurement are related to respective standard curves prepared withrespective precipitating reagents, and the quantity of soluble fibrinpresent in the opaque specimen sample is determined by an average of thetwo measurements.
 7. The method of claim 5, wherein the the firstprecipitating reagent and the second precipitating reagent are twoconcentrations of an identical precipitating reagent.
 8. The method ofclaim 5, wherein the first precipitating reagent and the secondprecipitating reagent are different.
 9. The method of claim 1, whereinthe opaque specimen fluid is an opaque body fluid from human containingsoluble fibrin.
 10. The method of claim 1, wherein the opaque specimenis selected from a group consisting of whole blood, bloody effusions,bloody cerebrospinal fluid, and the like.
 11. The method of claim 10,wherein the opaque specimen fluid is a diluted whole blood.
 12. Themethod of claim I wherein the precipitating reagent is protamine sulfateor polybrene.
 13. The method of claim 1, wherein the portion of theopaque specimen fluid is mixted with the precipitating reagent at a PHof about about 5.9 or below and a temperature of about 37° C.
 14. Themethod of claim 7, wherein the precipitating reagents are protaminesulfate or polybrene.
 15. The method of claim 8, wherein the firstprecipitating reagent is protamine sulfate, and the second precipitatingreagent is polybrene.